Hyperglycemia and increased transforming growth factor-? (TGF?) expression activate mTOR (mechanistic target of rapamycin), which acts as a key node to promote glomerular and tubular hypertrophy and matrix protein fibronectin expression. We have reported that high glucose and TGF? activate both mTOR complexes (C1 and C2) in mesangial and proximal tubular epithelial (PTE) cells and in kidneys of type 2 and type 1 diabetic mice. Blocking mTOR activity with rapamycin, we showed reduction in albuminuria, renal hypertrophy and matrix expansion in these models of diabetes. Complete inhibition of mTOR by rapamycin can be detrimental as the activity of this kinase is required for maintaining renal cell homeostasis. Also, rapamycin changes gene expression profile, which causes adverse effects. Therefore, alternative therapeutic approach is necessary to block mTOR kinase. Our data demonstrate that the expression of deptor, a component of both mTOR complexes, and inhibitor of their kinase activities, is reduced in kidneys of diabetic rodents and in mesangial and PTE cells treated with high glucose or TGF?. Exploiting how deptor is suppressed to increase mTOR activity can identify novel molecular targets to block diabetic renal complications. In our preliminary data, we find reduction in deptor levels concomitant with increased expression of EZH2 (enhancer of zeste homolg 2), the catalytic subunit of the polycomb repressor complex 2, which trimethylates histone H3 at lysine- 27 to block transcription of a specific gene. Moreover, we show enhanced expression of the E3 ubiquitin ligase ?TrCP (transducin repeat containing protein) by high glucose and TGF? in renal cells. ?TrCP targets deptor for degradation. Furthermore, we demonstrate high glucose and TGF? increase the expression of a family of microRNA, miR-181 (a,b,c,d), and miR-221. These microRNAs are also increased in kidneys of type 1 and type 2 diabetic mice and target deptor for downregulation. In this proposal, using cultured mesangial and PTE cells and renal tissues from diabetic OVE26 and db/db mice, we will test the hypothesis that inappropriate deptor downregulation contributes to renal hypertrophy and matrix expansion in diabetic kidney disease. Probing the specificities of underlying mechanisms, we will define the regulatory modules of deptor suppression. In the first specific aim, we will investigate EZH2 as a candidate for transcriptional suppression of deptor, hypertrophy and, fibronectin and PAI-1 (plasminogen activator inhibitor-1) expression. In the second aim, the role of ?TrCP in deptor protein degradation/mTORC1/C2 activation and its cross talk with TGF? signaling in forcing mesangial and PTE cell hypertrophy and, fibronectin and PAI-1 expression will be examined.
In specific aim 3, we will investigate the contribution of miR-181 family and miR-221 to hypertrophy and, fibronectin and PAI-1 expression in response to high glucose and TGF? in mesangial and PTE cells and in diabetic mice kidneys. To address these aims, techniques including immunoblotting, immunoprecipitation, qRT-PCR, morphometry, immunohistochemistry, reporter transfection assays, chromatin immunoprecipitation assays and siRNA-mediated downregulation of proteins will be used.

Public Health Relevance

A recent study showed that 29% of the hospitalized veterans had chronic kidney disease, which often leads to end stage renal disease (ESRD). Diabetes is one of the most prevalent chronic diseases among people aged 20 years and older in the US. The demographics of veteran population fall in this age group. 23.1% of people in the age group of 60 years or older have diabetes. Diabetic kidney disease is the leading cause of ESRD, accounting for about 44% of new cases each year. Current therapies only delay the progression to ESRD and do not arrest the disease.Thus there is an urgent need for therapeutic approaches, which may include promoting protective or regenerative factors and blocking mechanisms of injury. Renal cell hypertrophy and matrix expansion are two pathological features of diabetic kidney disease. This proposal will explore novel signaling protein, deptor, which can be targeted using small moleucule inhibitors and anti- microRNAs in the affected patients, who suffer from kidney disease resulting from complications of diabetes.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
5I01BX000926-08
Application #
9685029
Study Section
Nephrology (NEPH)
Project Start
2011-07-01
Project End
2020-06-30
Budget Start
2019-07-01
Budget End
2020-06-30
Support Year
8
Fiscal Year
2019
Total Cost
Indirect Cost
Name
South Texas Veterans Health Care System
Department
Type
DUNS #
078493228
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Das, Falguni; Ghosh-Choudhury, Nandini; Lee, Doug Yoon et al. (2018) Akt2 causes TGF?-induced deptor downregulation facilitating mTOR to drive podocyte hypertrophy and matrix protein expression. PLoS One 13:e0207285
Shi, Qian; Viswanadhapalli, Suryavathi; Friedrichs, William E et al. (2018) Nox4 is a Target for Tuberin Deficiency Syndrome. Sci Rep 8:3781
Das, Falguni; Ghosh-Choudhury, Nandini; Kasinath, Balakuntalam S et al. (2018) Tyrosines-740/751 of PDGFR? contribute to the activation of Akt/Hif1?/TGF? nexus to drive high glucose-induced glomerular mesangial cell hypertrophy. Cell Signal 42:44-53
Maity, Soumya; Bera, Amit; Ghosh-Choudhury, Nandini et al. (2018) microRNA-181a downregulates deptor for TGF?-induced glomerular mesangial cell hypertrophy and matrix protein expression. Exp Cell Res 364:5-15
Lee, Hak Joo; Lee, Doug Yoon; Mariappan, Meenalakshmi M et al. (2017) Hydrogen sulfide inhibits high glucose-induced NADPH oxidase 4 expression and matrix increase by recruiting inducible nitric oxide synthase in kidney proximal tubular epithelial cells. J Biol Chem 292:5665-5675
Das, Falguni; Ghosh-Choudhury, Nandini; Venkatesan, Balachandar et al. (2017) PDGF receptor-? uses Akt/mTORC1 signaling node to promote high glucose-induced renal proximal tubular cell collagen I (?2) expression. Am J Physiol Renal Physiol 313:F291-F307
Bera, Amit; Das, Falguni; Ghosh-Choudhury, Nandini et al. (2017) Reciprocal regulation of miR-214 and PTEN by high glucose regulates renal glomerular mesangial and proximal tubular epithelial cell hypertrophy and matrix expansion. Am J Physiol Cell Physiol 313:C430-C447
Das, Falguni; Dey, Nirmalya; Bera, Amit et al. (2016) MicroRNA-214 Reduces Insulin-like Growth Factor-1 (IGF-1) Receptor Expression and Downstream mTORC1 Signaling in Renal Carcinoma Cells. J Biol Chem 291:14662-76
Sataranatarajan, Kavithalakshmi; Ikeno, Yuji; Bokov, Alex et al. (2016) Rapamycin Increases Mortality in db/db Mice, a Mouse Model of Type 2 Diabetes. J Gerontol A Biol Sci Med Sci 71:850-7
Dey, Nirmalya; Bera, Amit; Das, Falguni et al. (2015) High glucose enhances microRNA-26a to activate mTORC1 for mesangial cell hypertrophy and matrix protein expression. Cell Signal 27:1276-85

Showing the most recent 10 out of 12 publications